The present invention, in some embodiments thereof, relates to fluorogenic compounds and, more particularly, but not exclusively, to chemically-activatable fluorogenic compounds which upon a chemical event rearrange so as produce a near infrared probe, and to uses thereof.
The use of non-radioactive labels in biochemistry and molecular biology has grown exponentially in recent years. Among the various compounds used as non-radioactive labels, aromatic dyes that produce fluorescent or luminescent signal are especially useful.
Near-infrared spectroscopy (NIRS) is a spectroscopic method that uses the near-infrared region of the electromagnetic spectrum (from about 700 nm to 1400 nm). NIR can typically penetrate much farther into a sample than mid infrared radiation, and is particularly useful in probing bulk material with little or no sample preparation. Optical imaging in a near-infrared (NIR) range of between 650 nm and 900 nm is highly suitable for biological applications since it enables detection of molecular activity in vivo due to high penetration of NIR photons through organic tissues and low autofluorescence background at this range.
Fluorochromes suitable for use in NIRS include, for example, fluorescein labels, Alexa Fluor dyes, cyanine dyes such as Cy2, Cy3, Cy5, Cy7, optionally substituted coumarin, R-phycoerythrin, allophycoerythrin, Texas Red and Princeston Red.
Cyanine dyes have been recognized as useful for NIR imaging, due to the biological compatibility, environmental stability, the large extinction coefficient, narrow emission bands and relatively high quantum yield thereof [Mujumdar ET AL. Bioconjugate Chem. 4, 105-11 (1993)], which allow them to penetrate deep tissues and serve as attractive probes for intravital non-invasive imaging.
The cyanine dyes are comprised of two nitrogen-containing moieties (e.g., indolenine based rings), one being in a form of an acceptor moiety (e.g., ammonium) and one in the form of a donor moiety (e.g., amine), which are connected by a series of conjugated double bonds. The dyes are classified by the number (n) of central double bonds connecting the two ring structures; monocarbocyanine or trimethinecarbocyanine when n=1; dicarbocyanine or pentamethinecarbocyanine when n=2; and tricarbocyanine or heptamethinecarbocyanine when n=3.
Cyanine dyes have been developed for use in Fluorescence Resonance Energy Transfer (FRET) assays. Briefly, FRET assays depend on an interaction between two fluorophores, a donor fluorophore and an acceptor fluorophore. When the donor and acceptor molecules are in close enough proximity, the fluorescence of the donor molecule is transferred to the acceptor molecule with a resultant decrease in the lifetime and a quenching of fluorescence of the donor species and a concomitant increase in the fluorescence intensity of the acceptor species. In the case of peptide cleavage reactions, a fluorescent donor molecule and a fluorescent acceptor molecule are attached to a peptide substrate on either side of the peptide bond to be cleaved and at such a distance that energy transfer takes place. A peptide cleavage reaction will separate the donor and acceptor molecules and thus the fluorescence of the donor molecule will be restored.
Cyanine dyes suitable for use as acceptor or “quencher” molecules in a FRET assay have been developed by making certain modifications to cyanine dyes through introduction of chemical groups which have the effect of diminishing or abolishing the fluorescence of the molecule.
For example, a Turn-ON system for a cyanine molecule which utilizes FRET comprises a cyanine dye and a quencher attached to one another through an enzymatically-cleavable linker, to obtain a quenched fluorophore. Once the linker is cleaved by a specific enzyme, the fluorophore separates from the quencher and thus, turn-ON of a fluorescence signal is effected. This approach, however, requires an additional dye (the quencher) apart from the cyanine.
Efforts to design alternative Turn-ON cyanine probes typically had limitations of low quantum yield, low extinction coefficient and complex synthesis [see, for example, Ho et al. Bioorg. Med. Chem. Lett. 16, 2599-602 (2006); and Richard et al. Org. Lett. 10, 4175-8 (2008)], and only limited specific examples for cyanine probes that involve changes with the π-electrons conjugation have been reported [see, Kundu et al. Angew. Chem. Int. Ed. 49, 6134-8 (2010); and Kundu et al. Angew. Chem. Int. Ed. 48, 299-303 (2009)].
Cyanine molecules coupled to targeting peptides have been reported. See, for example, Tung et al., Bioconjug Chem. 1999 10(5):892-6, which discloses a cathepsin-sensitive system that comprises a cyanine-peptide conjugate; and U.S. Pat. No. 6,217,848, which disclosed dye-peptide conjugates, including several cyanine dyes with a variety of bis- and tetrakis (carboxylic acid) homologues, for targeted delivery.
Additional background art includes Samanta et al. Chem. Commun., 2010, 46, 7406-7408; Dickinson et al. J. Am. Chem. Soc. 132, 5906-15 (2010); Dickinson et al. Nat. Chem. Biol. 7, 106-12 (2011); Miller et al. Nat. Chem. Biol. 3, 263-7 (2007); Van de Bittner et al. Proc. Natl. Acad. Sci. (2010)]; Avital-Shmilovici, M. & Shabat, D. Biorg. Med. Chem. 18, 3643-7 (2010); Sella, E. & Shabat, D. Chem. Commun., 5701-3 (2008); Sella, E. & Shabat, D. J. Am. Chem. Soc. 131, 9934-6 (2009); Wainstein et al., Chem. Commun. 2010; 46: 553-5; Karton-Lifshin et al., J. Phys. Chem. A. 2012 Jan. 12; 116(1):85-92; Karton-Lifshin et al., J. Am. Chem. Soc. 2011 Jul. 20; 133(28):10960-5; and Sella et al., J. Am. Chem. Soc., 132, 3945-3952.